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European Journal of Cell Biology

formerly C Y T O B I O L O G I E

Volume 24 Number 1 April 1981

W I S S E N S C H A F T L I C H E V E R L A G S G E S E L L S C H A F T M B H

Adolph, Κ. W.: A serial sectioning study of the structure of human mitotic chromosomes 146

Ammermann, D., G. Steinbrück, R. Baur, H . Wohlert (Short Communication): Methylated bases in the D N A of the ciliate Stylonychia mytilus 154

Arnold, C.-G., see Blank, R. 244

Augeron, C , see Daudet, F. 197

Avramova, Ζ., see Tsanev, R. 139

Baumgartner, Β., see Herman, Ε. M . 226

Baur, R., see Ammermann, D . 154

Behnke, O., see Tranum-Jensen, J. 275

Behnke, O., see Tranum-Jensen, J. 281

Bernaert, D., see Wanson, J.-C. 88

Bignami, Α., see Dahl, D. 191

Bilinski, Μ., H . Plattner, R. Tiggemann: Isolation of surface membranes from normal and exocytotic mutant strains of Pa­ramecium tetraurelia. Ultrastructural and biochemical char­acterization 108

Blank, R., C.-G. Arnold: Structural changes of mitochondria in Chlamydomonas reinhardii after chloramphenicol treat­ment 244

Boothroyd, B., see Hopkins, C. R. 259

Brown, D. L. , see Rogers, Κ. A. 1

Chemnitz, J., see Junker, St. 16

Chrispeels, Μ. J., see Herman, Ε. M . 226

Christiansen, Ε. N . , see Fiatmark, T. 62

Dahl, D., D. C. Rueger, A. Bignami, K. Weber, M . Osborn: Vimentin, the 57000 molecular weight protein of fibroblast filaments, is the major cytoskeletal component in immature glia 191

Daudet, F., C. Augeron, Μ. Ollivier-Bousquet: Effet rapide in vitro de la colchicine, du chlorure d'ammonium et de la prolactine, sur la secretion des lipides du lait dans la glande mammaire. (Early action of colchicine, ammonium chloride, and prolactin, on secretion of milk lipids in the lactating mammary gland.) 197

Debus, Ε., K. Weber, M . Osborn: The cytoskeleton of blood platelets viewed by immunofluorescence microscopy 45

Ellinger, Α., see Pavelka, M . 53

Fakan, J., see The Can Ton-That 317

Feierabend, J., H . Grunz, Η. W. Sauer (Meeting report): Regulation of differentiation in eukaryotic model systems 157

Flatmark, Τ., Ε. N . Christiansen, H . Kryvi : Polydispersity of rat liver peroxisomes induced by the hypolipidemic and car­cinogenic agent Clofibrate 62

Franke, W. W., see Spring, H . 298

Gautier, Α., see The Can Ton-That 317

Gerbes, A. L., see Nees, S. 287

Gerlach, E., see Nees, S. 287

Gicquaud, C , A. Loranger (Short Communication): La phal-loidine protege la F-actine contre les effets destructeurs de Facide osmique. I I . Protection par la phalloidine de la F-ac­tine traitee aux aldehydes. (Phalloidin counteracts the de­struction of F-actin by osmic acid. I I . Protection by phalloid­i n of F-actin crosslinked with aldehydes.) 320

Gotlieb, A . I . , see Kalnins, V. I . 36

Gr^becki, Α.: Effects o f localized photic stimulation on amoeboid movement and their theoretical implications 163

Gregory, H. , see Hopkins, C. R. 259

Groot, C. de, J. Wormmeester: Capping of surface immuno­globulin on rabbit and mouse lymphocytes. I . Kinetics 9

Grunz, Η., see Feierabend, J. 157

Hackenbrock, Ch. R., see Sowers, A. E. 101

Herman, Ε. Μ., B. Baumgartner, Μ. J. Chrispeels: Uptake and apparent digestion o f cytoplasmic organelles by protein bodies (protein storage vacuoles) in mung bean cotyledons 226

Herzog, V., F. Miller: Structural and functional polarity of inside-out follicles prepared from pig thyroid gland 74

Herzog, V., see Schmidt, H.-W. 85

Hopkins, C. R., B. Boothroyd, H . Gregory: Early events fol­lowing the binding o f epidermal growth factor to surface re­ceptors on ovarian granulosa cells 259

Junker, St., J. Chemnitz: Ultrastructural characterization of a mouse melanoma cell line, a mouse fibroblastic cell line, and hybrids between them 16

Kalnins, V. L, L . Subrahmanyan, A. I . Gotlieb: The reorgan­ization o f cytoskeletal fibre systems in spreading porcine en­dothelial cells in culture 36

Khoshbaf, Μ. Α., see Rogers, Κ. A. 1

Kochert, G. D., see Pommerville, J. C 236

Koller, Th. , see Labhart, P. 309

Kryv i , H . , see Flatmark, T. 62

Kurelec, B., see Müller, W. E. G. 28

Labhart, P., Th . Koller: Electron microscope specimen pre­paration of rat liver chromatin by a modified Miller spread­ing technique 309

Loranger, Α., see Gicquaud, C. 320

Maihotra, S. K., see Sikerwar, S. S. 211

Maslov, O., see Ogievetskaya, Μ. M . 124

Maurer, Α., Κ. Mühlethaler: Isolation and characterization of paracrystalline arrays o f the plasma membrane of baker's yeast Saccharomyces cerevisiae 216

Meinzer, P., see Schroeter, D . 131

Metenier, G.: Chronologie de la Synthese du D N A macro-nucleaire chez les formes predatrices de Tetrahymena para-vorax. (Timing o f macronuclear D N A synthesis in carnivor­ous forms of Tetrahymena paravorax.) 252

Miller, F., see Herzog, V. 74

Miller, F., see Schmidt, H.-W. 85

Miquelis, R., C. Simon: The thyroid lysosomal system: dy­namic state of the organelles in relation to iodine release 70

Mühlethaler , Κ., see Maurer, Α. 216

Müller, I . , see Müller, W. Ε. G. 28

Müller, W. Ε. G., R. Κ. Zahn, I . Müller, Β. Kurelec, G. Uh-lenbruck, P. Vaith: Cell aggregation of the marine sponge Geodia cydonium. Identification of lectin-producing cells 28

Nees, S., A. L . Gerbes, E. Gerlach, J. Staubesand: Isolation, identification, and continuous culture of coronary endothe­lial cells from guinea pig hearts 287

I I I

Ogievetskaya, Μ. Μ., Ο. Maslov, Ν. Sheludko: Trace ele­ments in phasic and tonic muscles 124

Ollivier-Bousquet, M . , see Daudet, F. 197

Osborn, Μ., see Dahl, D. 191

Osborn, M , see Debus, E. 45

Osborn, M . , see Shaw, G. 20

Pavelka, M . , A. Ellinger: Effect of colchicine on the Golgi ap­paratus and on GERL of rat jejunal absorptive cells. Ultra-structural localization of thiamine pyrophosphatase and acid phosphatase activity 53

Pebusque, M.-J., A. Robaglia, R. Seite: Diurnal rhythm of nucleolar volume in sympathetic neurons of the rat superior cervical ganglion 128

Penasse, W., see Wanson, J.-C. 88

Piwowar, U., see Strzelecka-Golaszewska, H. 116

Plattner, Η., see Bilinski, M . 108

Plattner, Η., see Tiggemann, R. 184

Pliszka, B., see Strzelecka-Golaszewska, H . 116

Pommerville, J. C , G. D. Kochert: Changes in somatic cell structure during senescence of Volvox carteri 236

Popowski, Α., see Wanson, J.-C. 88

Rassat, J., H . Robenek, H. Themann: Ultrastructural changes in mouse hepatocytes exposed to vinblastine sulfate with spe­cial reference to the intercellular junctions 203

Robaglia, Α., see Pebusque, M.-J. 128

Robenek, H., see Rassat, J. 203

Rogers, Κ. Α., Μ. A. Khoshbaf, D. L . Brown: Relationship of microtubule organization in lymphocytes to the capping of immunoglobulin 1

Rueger, D. C , see Dahl, D. 191

Sauer, Η. W., see Feierabend, J. 157

Schmidt, H.-W., V. Herzog, F. Miller: Interaction of carba-chol and isoproterenol on 0 2 consumption and morphology of isolated acini from rat parotid gland 85

Schroeter, D., D. Werner, P. Meinzer: Size of native and de­natured D N A of Ehrlich ascites tumour cells isolated in the presence of different protease concentrations 131

Schultz, J. E., see Walter, M . F. 97

Seite, R., see Pebusque, M.-J. 128

Shaw, G., M . Osborn, K. Weber: Arrangement of neurofila­ments, microtubules, and microfilament-associated proteins in cultured dorsal root ganglia cells 20

Sheludko, N . , see Ogievetskaya, Μ. M . 124

Sikerwar, S. S., J. P. Tewari, S. K . Malhotra: Subunit struc­ture of the connexons in hepatocyte gap junctions 211

Simon, C , see Miquelis, R. 70

Sowers, A. E., Ch. R. Hackenbrock: Alterations in density and size distribution of intramembrane particles in the inner membrane of mitochondria from chloramphenicol-fed mice 101

Spring, H., W. W. Franke: Transcriptionally active chroma­tin in loops of lampbrush chromosomes at physiological salt concentrations as revealed by electron microscopy of sec­tions 298

Staubesand, J., see Nees, S. 287

Steinbrück, G., see Ammermann, D. 1 5 4

Strzelecka-Golaszewska, H., U . Piwowar, B. Pliszka: Changes in the ultrastructure of actomyosin gel during hy­drolysis of ATP under various ionic conditions 116

Subrahmanyan, L., see Kalnins, V. I . 36

Tewari, J. P., see Sikerwar, S. S. 211

The Can Ton-That, G. Turian, J. Fakan, A. Gautier (Short Communication): Ultrastructural cytochemistry of perinu­cleolar dense spots in heat-treated macroconidia of Neuros-pora crassa 317

Themann, Η., see Rassat, J. 203

Tiggemann, R., see Bilinski, M . 108

Tiggemann, R., H . Plattner: Localization of actin in the cor­tex of Paramecium tetraurelia cells by immuno- and affinity-fluorescence microscopy 184

Tranum-Jensen, J., O. Behnke: Acetylcholinesterase in the platelet-megakaryocyte system. I . Structural localization in platelets of the rat, mouse, and cat 275

Tranum-Jensen, J., O. Behnke: Acetylcholinesterase in the platelet-megakaryocyte system. I I . Structural localization in megakaryocytes of the rat, mouse, and cat 281

Tsanev, R., Z. Avramova: Nonprotamine nucleoprotein u l -trastructures in mature ram sperm nuclei 139

Turian, G., see The Can Ton-That 317

Turner, Β. M. : Isolation of monoclonal antibodies to chroma­tin and preliminary characterization of target antigens 266

Uhlenbruck, G., see Müller, W. E. G. 28

Vaith, P., see Müller, W. E. G. 28

Walter, M . F., J. E. Schultz: Calcium receptor protein calmo­dulin isolated from cilia and cells of Paramecium tetraurelia 97

Wanson, J.-C, W. Penasse, D. Bernaert, A. Popowski: Glu-cose-6-phosphatase distribution in isolated and cultured adult rat hepatocytes 88

Weber, K., see Dahl, D. 191

Weber, K., see Debus, E. 45

Weber, K., see Shaw, G. 20

Weber, K., see Wehland, J. 176

Wehland, J., K . Weber: Actin rearrangement in living cells revealed by microinjection of a fluorescent phalloidin deriva­tive 176

Werner, D., see Schroeter, D. 131

Wohlert, H. , see Ammermann, D. 154

Wormmeester, J., see Groot, C. de 9

Zahn, R. Κ., see Müller, W. Ε. G. 28

Annual meeting of the Deutsche Gesellschaft fur Zellbiolo-gie, Deutsche Gesellschaft für Genetik, and Gesellschaft für Entwicklungsbiologie, Freiburg, 26-28 March 1981 (Ab­stracts) 324

Coming Events 160

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European Journal of Cell Biology 24, 287-297 (1981) © Wissenschaftliche Verlagsgesellschaft mbH · Stuttgart

Isolation, identification, and continuous culture of coronary endothelial cells from guinea pig hearts

S. Nees1), A. L . Gerbes, E . Gerlach

Department of Physiology, University of Munich/Federal Republic of Germany

J. Staubesand Department of Anatomy, University of Freiburg i . Br./Federal Republic of Germany

Received February 9, 1981 Accepted March 11, 1981

Coronary endothelial cells—endothelial cell culture — 5'-nucleo­tidase— adenosine — adenine nucleotide metabolism — isolation of endothelial cells

Viable and homogeneous endothelial cells were obtained from iso­lated guinea pig hearts by application of a special perfusion tech­nique o f the coronary system with an isotonic collagenase-trypsin so­lution and subsequent purification of the dissociated cells by Percoll density gradient centrifugation. The coronary endothelial cells were grown in tissue culture for periods up to 7 months. Serial passage proved to be possible. During logarithmic growth, generation time was found to be 18 h; it could be reduced to 16 h by addition of thrombin to the culture medium. Light, phase contrast and scanning electron microscopy as well as autoradiography revealed that cul­tured coronary endothelial cells grew as strict monolayers of closely apposed, polygonal large cells. By scanning electron microscopy, it could be demonstrated that the morphology of the cultured cells changed characteristically during attachment of the cells to their sub­stratum. The changes observed were very similar to those of prolifer­ating endothelial cells of isolated coronary vessels kept in organ cul­ture. According to transmission electron microscopy studies, cultured coronary endothelial cells proved to contain only an extremely small number o f Weibel-Palade bodies. Nucleoside Phosphorylase (EC 2.4.2.5.) and 5'-nucleotidase (EC 3.1.3.5.) were identified in freshly isolated as well as in cultured endothelial cells. Their specific and to­tal activities proved to be much higher than in myocardial tissue, thus indicating a prominent role of nucleotide metabolism in the cor­onary endothelium.

Introduction

Endothelial cells are known to be involved in the regulation of transport processes across vessel walls [35, 6] and in he­mostatic mechanisms [22, 23]. Structural, metabolic and functional abnormalities of the endothelium may be of parti­cular significance in certain disorders, such as atherosclerosis [43, 44], diabetic angiopathy [9], inflammatory processes [54] and thrombosis [19, 20]. In addition, there is increasing evi-

' ) Dr. S. Nees, Physiologisches Institut der Universität, Pettenko-ferstr. 12, D-8000 München 2/Federal Republic of Germany.

dence that endothelial cells can participate in the local con­trol of circulation by means of secretion [39], binding [7] or inactivation [25] of vasoactive metabolites and by their as­sumed ability to contract spontaneously [32, 18, 2].

Endothelial cell metabolism and its regulation is poorly understood, mainly because of methodological problems which hinder or exclude biochemical analyses of intact endo­thelial cells under in situ conditions. On the other hand, it has now become possible to study biochemical features of endothelial cells grown in culture under physiologically de­fined and carefully controlled conditions. So far successful cultivation has been reported for endothelial cells derived from human umbilical veins [12, 24], bovine vena cava [55], the portal vein of guinea pigs [4], the bovine pulmonary ar­tery [8], and aortae from cattle [21], rabbits [30] and pigs [52]. To our knowledge, there is no report in the literature on cul­ture and long term maintenance of identified endothelial cells from the circulatory system of isolated organs.

This paper describes a procedure for the isolation and cul­tivation of homogeneous coronary endothelial cells from guinea pig hearts. The cultured cells were characterized by means of light and electron microscopic techniques, by their growth behaviour and their generation times. In addition, we have determined the specific activities of two enzymes which have been previously shown to be characteristic for coronary endothelial cells in guinea pigs [46, 47], Preliminary results from these investigations have been reported at the 51st Con­gress of the German Physiological Society [36] and at the 3rd International Symposium on "Purine Metabolism in Man" [37].

Materials and methods

Enzymes, chemicals, reagents, and media

Trypsin, coliagenase (CI. histolyticum, type I I ) , medium 199, fetal calf serum (FCS), glutamine, penicillin and streptomycin were pur­chased from Seromed, München. Sodium salts of 5 -AMP and α-gly­cerophosphate, inosine and xanthine oxidase were obtained from Boehringer, Mannheim. Pure and highly polymerized D N A (calf

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Tab. I . Specific and total activities of 5'-nucleotidase and nucleoside Phosphorylase in freshly isolated and cultured coronary endothelial cells (secondary culture) and in ventricular myocardium of isolated perfused guinea pig hearts. Data are means from separate analyses of three series of culture dishes and 3 hearts.

Specific activity Total activity [nmoles/min χ mg protein] [nmoles/min χ g wet tissue]

Endothelial cells Myocardium Endothelial cells Myocardium isolated cultured isolated cultured

5'-Nucleotidase 77 75 4 19 300 19 100 1 880 Purine nucleoside Phosphorylase 232 234 3 37 000 37 400 760

thymus) was supplied by Sigma, St. Louis, USA. (Methyl- , 4 C)-thy-midine (53 mCi/mmol) and (methyl- 3H)-thymidine (42 Ci /mmol) were purchased from Amersham-Buchler, England. Reagents for fix­ation and embedding tissue samples and cells prior to electron micro­scopy (glutaraldehyde; dodecenyl succinic acid anhydride; osmium tetroxide; Epon 812; 2,4,6-tri(dimethyl-aminomethyl)phenol; methyl-nadic anhydride) and the phosphate determination kit were obtained from Serva, Heidelberg. Photoemulsion NTB-2 for autoradiography was purchased from Eastman Kodak Corp., Rochester, Ν. Y. Percoll and Cytodex were obtained from Pharmacia, Uppsala. The protein test kit was supplied by Biorad (Richmond, Cal.). A l l other chemi­cals of the highest available purity were purchased from Merck, Darmstadt.

Culture medium. Medium 199 was supplemented with 20% FCS, 200 U / m l penicillin, 20 μ g / m l streptomycin and 2 m M glutamine.

Phosphate buffered saline (PBS). 140 m M NaCl, 4 m M K C l and 1 mM potassium phosphate buffer, pH 7.4.

Fig. 1. Schematic representation of steps involved in the perfusion of the guinea pig heart and in the collection of dissociated coronary endothelial cells. The heart is completely immersed into culture me­dium containing 20% sucrose. The coronary endothelial cells are en-zymatically detached by perfusion of the coronary system with iso­tonic collagenase-trypsin solution (aortic valves are closed). The per­fusate containing the endothelial cells, overlayers the sucrose solu­tion, thus preventing contact of the heart surface with the en­zymes.— 1 Cannula. — 2 Aorta. — 3a Left, 3b right coronary ar­tery.—4 Coronary sinus. — Arrows direction of perfusion. — Ε Endo­thelial cell layer. — Μ Medium Μ 199 containing sucrose (20% w/v) .

Percoll gradients. 70 ml Percoll and 30 ml o f 3% (w/v ) NaCl were aseptically mixed and centrifuged at 30000 χ g for 1 h in plastic cen­trifuge tubes in a Sorvall centrifuge RC2-B equipped with a SS-34 rotor.

Isolation of coronary endothelial cells

A l l procedures were performed aseptically. Hearts from ether-anaes­thetized female guinea pigs (250-300 g) were isolated and perfused by means of a peristaltic pump according to the technique of Lan-gendorff [31]. In order to obtain a non-beating preparation part of the right atrium was dissected. After an initial perfusion with PBS for 5 min (constant volume, 5 ml /min , 40 °C) the cannulated hearts, still connected to the pump, were completely immersed into 50 ml conical tubes (Falcon Plastics, Oxnard, Cal.) filled wi th 20 ml medium 199 containing 20% sucrose (w/v) at 40 °C (Fig. 1). Thus contact was minimized between heart surface and enzyme solution used to dis­lodge the coronary endothelial cells.

Dispersion of the endothelial lining of the coronary vessels was ini ­tiated by a perfusion period of 1 min at 40 °C with dissociation me­dium (PBS containing 0.1% collagenase (w/v) and 0.1% trypsin (w/ v)). The perfusate leaving the coronary sinus within this time over-layered immediately the more dense sucrose immersion medium. After ending the initial perfusion, the coronary system was exposed for 15 min to the enzyme solution which had been retained in the vascular space. Endothelial cells which had been dissociated from the vessel walls during the exposure to the enzymes, were then flushed out of the heart by a short perfusion with PBS devoid of enzymes. The concentrated cell suspension combined with the enzyme solution which already overlayered the sucrose medium. This crude endothe­lial cell harvest was aspirated and centrifuged (10 min, 250 g). The sedimented cells were washed with culture medium and centrifuged once again.

Washed cells harvested from 10 hearts were combined and sus­pended in 2 ml of fresh culture medium, which was then transferred to the top of a Percoll gradient [41]. A subsequent low speed centrifu-gation (30 min, 1000 g) resulted in the separation of endothelial cells

Fig. 2. Selective detachment of coronary endothelial cells. Photo­graphs a and b were taken after perfusion of the coronary system with isotonic buffer. The pictures c and d were taken after perfusion of the coronary system with isotonic collagenase-trypsin solution (0.1% w/v, each). — a. Scanning electron micrograph of the luminal side of a coronary artery. The endothelial sheet built up by closely arranged individual cells is intact.— 1800 χ . — b. Transmission elec­tron micrograph of a capillary demonstrating good preservation of the microvasculature. — L Capillary lumen. — Ν Nucleus of a capil­lary cell. — Μ Myocardium. —16000 χ . — c. Scanning electron mi­crograph of a coronary artery which reveals a complete desquama­tion of the endothelium. Arrows show remaining laminal structures of the intima. — 1250x. — d. Transmission electron micrograph de­monstrating efficient dissociation of a capillary. The capillary space (L) within the myocardium (M) is devoid of endothelial cells. Arrow­heads indicate parts of the basal lamina. — 12000 χ .

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from contaminating cells (mainly erythrocytes) and from some cell debris. The uppermost layer in the tubes contained a homogenous endothelial cell suspension. This layer was aspirated and diluted to 50 ml with culture medium. After a final centrifugation (10 min, 250 g) the purified cell harvest was resuspended in 80 ml of culture me­dium. This suspension contained approximately 2.5 χ 104 cells/ml.

Culture conditions and subcultivation

Seeding. The final suspension of coronary endothelial cells was dis­tributed equally among 40 Petri dishes (Falcon Plastics 3001, 0 35 mm). Cultivation was performed at 40 °C in a water saturated atmo­sphere containing 3% C 0 2 . After 24 h and then weekly the culture medium was replaced by a freshly prepared solution.

Secondary passage of cells. Primary cell cultures microscopically controlled and selected for subcultivation were washed with PBS and treated for 15 min at 40 °C with the dissociation medium. A complete detachment of all cells was facilitated by use of a sterile PVC scraper. The suspended cells were then collected by centrifuga­tion (10 min, 250 g) and washed twice with culture medium. Seeding and cultivation was performed as described above. Generation times were calculated according to standard procedures. Cell densities were daily documented by photomicrography using an inverted micro­scope (Leitz Μ 3 camera).

Microbead cultivation. 100 mg of Cytodex dextran beads (mean di­ameter about 100 μηι) were sterilized and washed according to the instructions of the manufacturer. After centrifugation the beads were resuspended in 10 ml of culture medium, which then was distributed among 5 standard culture dishes. Inoculation with about 105 cells and incubation for 1 week under the conditions described above re­sulted in the formation of endothelial monolayers covering each bead. They were used for ultrastructural studies by means of trans­mission electron microscopy.

Morphological characterization

Silver impregnation. Cultures were rinsed twice with PBS and twice with 5% glucose. Intercellular borders were specifically stained with 0.25% silver nitrate solution and fixed in 9% formalin as described by Garbarsch et al. [13].

Scanning electron microscopy (SEM). Guinea pig hearts automati­cally beating were first perfused for 10 min at constant pressure (80 mm Hg) with PBS containing 6% albumin, and then for 60 min with 2.5% glutaraldehyde solution in 90 m M sodium cacodylate buffer (pH 7.4) containing 0.5 m M CaCl 2 .

0 10 20 30 Fract ion

Fig. 3. Purification of a crude endothelial cell harvest by Percoll density centrifugation. — Left panel: Centrifuge tube with the respec­tive positions of the separated cells and tissue debris. — Right panel: Buoyant density of fractions collected from the gradients. — I Pure coronary endothelial cells. — I I - I V Contaminating cells and tissue debris, erythrocytes being exclusively present in band I I I .

Cultured endothelial cells and coronary arteries aseptically dis­sected and kept in organ culture for several days under the condi­tions described as well as freshly prepared heart slices (thickness 3 mm) were rinsed with PBS and prefixed in glutaraldehyde solution. The specimens were washed three times with cacodylate buffer con­taining 5% sucrose and then postfixed in 1% osmium tetroxide dis­solved in cacodylate buffer. The samples were then dehydrated in graded ethanol solutions (50-100%) and "critical point dried" in C 0 2

(37-40 °C; 739.6 N / c m 2 (73 Kp/cm 2 ) ) . Tissue samples were then at­tached to polished aluminium stubs, coated uniformly in vacuo with gold (thickness about 15 nm) and viewed immediately with a scan­ning electron microscope (Cambridge Instrument Co., Stereoscan).

Transmission electron microscopy (TEM). Samples of freshly iso­lated guinea pig hearts as well as coronary endothelial cells cultured on dextran microbeads (Cytodex, Pharmacia) were washed and fixed in glutaraldehyde and osmium tetroxide as described above. The samples were dehydrated in ethanol, stained en bloc with 0.5% ura-nyl acetate and 1% phosphotungstic acid in 75% ethanol, and embed­ded in Epon 812. Ultrathin sections were stained with uranyl acetate and lead citrate and viewed in a transmission electrone microscope (Zeiss, EM 9).

Analytical procedures

Protein content of PBS-washed cultures and of enzyme preparations were measured using a dye adsorption protein assay (Biorad, Rich­mond, Cal.). D N A was determined according to the diphenylamine color reaction of Ash well [1].

Preparation of enzyme extracts. Minced ventricular tissue and freshly isolated as well as cultured endothelial cells were suspended in 0.25 Μ Hepes buffer, pH 7.4, (about 2-10 mg tissue/ml, respective­ly) and disrupted by aid of a Dounce homogenizer. Ventricular ho-mogenates were centrifuged at 200 g for 10 min and the supernatants dialysed against 0.05 Μ Hepes buffer; pH 7.4. Endothelial cell lysates were not centrifuged, but dialysed directly under identical condi­tions.

Enzyme assays

Purine nucleoside Phosphorylase (EC 2.4.2.5.): A mixture of 100 μΐ inosine solution (10 m M ) , 400 μΐ potassium phosphate buffer (0.1 M , pH 7.4) and of 300 μΐ enzyme extract (containing up to 100 μg pro­tein) was incubated for 10-30 min at 37 °C. After boiling for 3 min, samples were cooled to room temperature and xanthine oxidase was added (about 0.04 U/20 μΐ). Hypoxanthine was determined by dif­ferential spectrophotometry after its enzymatic conversion to uric acid according to the method of Kalckar [26].

5'-nucleotidase (EC 3.1.3.5.): A reaction mixture of 100 μΐ A M P solution (24 m M ) , 50 μΐ MgCl 2 (0.1 M ) , 50 μΐ Hepes buffer (0.25 M , pH 8.4) and 50 μΐ enzyme extract (containing up to 400 μg protein) was incubated for 10-30 min at 37 °C. After heat denaturation of the enzyme (95 °C, 3 min) inorganic phosphate was determined accord­ing to Eibl [10] using a commercial phosphate determination kit (Ser-va). Values obtained were corrected for unspecific phosphate mono-esterase activity, which was determined under identical assay condi­tions, but using a-glycerolphosphate (24 m M ) instead of AMP-solu-tion.

Radioactive measurements

incorporation of 3 Η-thymidine into DNA. Secondary coronary endo­thelial cell cultures were exposed for 4 h to (methyl- 3H)thymidine (40-60 Ci /mmol , 1.0 μ Ο / π ι Ι culture medium). The radioactive me­dium was aspirated and each culture was extensively rinsed at 0 ° C with PBS. After fixation with acetic acid:ethanol (1:3 v /v) for 10 min, the samples were rinsed with distilled water, extracted with 10% (w/v) trichloracetic acid (TCA) for 10 min, followed by a second ex­traction with 5% TCA. After a final rinse with ethanol (96%) the cell layers were air dried and solubilized in 1 ml of 0.25 Μ NaOH by ul-

290

trasonification (Branson sonifier). Aliquots of the solution were counted in a Packard Tr i Carb Liquid scintillation spectrometer, model 3380. Each sample was made up to 10 ml with scintillation fluid (5.5 g of 2,5-diphenyloxazole (PPO) and 0.15 g of l,4-bis-2(-5-phenyloxazolyl)benzene (POPOP) in 666 ml of toluene and 323 ml of triton X 100. Counting efficiencies were 42 to 45% under these condi­tions.

Autoradiography. Coronary endothelial cell cultures at different growth stages were labelled with (methyl- 3H)-thymidine and washed as described above. The air dried cell layers on the bottom of their dishes were coated with Kodak NTB-2 emulsion and exposed for 3 days at 4 ° C . The counts of labelled nuclei were microscopically de­termined.

Results

Isolation of coronary endothelial cells

According to the endothelial classification of Bennett et al. [3] the coronary endothelium belongs to group Α- la , which is mainly characterized by a complete and continuous base­ment membrane and by lack of fenestrations or pores in the capillary system. This arrangement of the coronary endothe­lium as a uniform monolayer of flattened cells was not al­tered during perfusion for 4 min with PBS (Figs. 2a, b). Ex­posure of the coronary system for 15 min to collagenase-tryp­sin solution followed by PBS-perfusion, resulted in a specific detachment of the endothelial lining, both in large and small vessels (Figs. 2c, d). The extent of this enzymatically induced desquamation of the coronary endothelial cells was found to be dependent on the concentration and grade of the digestive enzymes, their contact time with the vessel walls and the temperature. Treatment for 60 min of the vessels resulted al­ready in a partial degradation of the vascular smooth muscle wall.

The endothelial cell harvest of usually 1 to 5 χ 105 viable cells from one heart was contaminated by small amounts of red cells and leukocytes, some large or spindle-shaped cells (apparently smooth muscle cells and fibroblasts), and some tissue debris most likely originating from the elastic lamina of the larger vessels. These contaminants (specific density 1.06-1.12 g/cm3) could easily be separated from the endothe­lial cells by Percoll density gradient centrifugation [41], Due to their strikingly low specific density of only 1.03 g/cm 3 the endothelial cells remained floating on top of the gradients (Fig. 3).

Growth and culture architecture

Within 3 h after seeding, 4096 of the freshly isolated endothe­lial cells characteristically clumped (Fig. 4a) started to flatten and spread out. The non-attached and apparently non-viable cells were removed from the culture dishes after 24 h with the first exchange of the medium. Convincing proof concern­ing growth and homogeneity at this stage of the culture was obtained by application of phase or interference contrast mi­croscopy. Within the first four days small colonies of typical architecture were formed (Fig. 4b), which gradually in­creased and coalesced within 3 to 5 weeks. The individual cells were homogeneous, closely apposed, polygonal ( 0 20-30 μΐη) with an oval, centrally located nucleus, a granular perinuclear region and undistinct cell borders (Fig. 4c). Cul-

Fig. 4. Three states of coronary endothelial cell growth in tissue culture. — a. Freshly isolated, characteristically clumped cells imme­diately after seeding. Light microscopy. — 300 χ . — b. A small group of endothelial cells 1 week after culture initiation. Interference con­trast microscopy ( N O M A R S K I optics). —520 χ . — c. Confluent en­dothelial monolayer after about 4 weeks. Phase contrast. — 600 χ .

ture architecture proved to be uniform and did not reveal multilayering of cells, a phenomenon commonly observed in cultures of fibroblasts or smooth muscle cells. Postconfluent cultures (up to 7 months after confluency) with a density of about 5 χ 105 cells/cm2 preserved the typical monolayer ar­rangement of individual cells. This could be documented by silver impregnation of the cultures, which revealed silver de­posits in one microscopic plane only along the cell borders.

291

days

Fig. 5. (Methyl- 3H)-thymidine incorporation into TCA-insoluble D N A ( A — A ) and growth curve of secondary coronary endothelial cells ( o — o ) . Data represent the mean from measurements in 4 sepa­rate culture dishes.

© 300

20 days

Fig. 6. D N A and protein content of coronary endothelial cells in secondary culture at different states of growth. — Starting cell density 2 χ 1G5 ceils per dish. — Density in postconfluent cultures 5.4 χ 106

cells per dish. Data are the average from measurements in 3 separate dishes.

Generation time

Coronary endothelial cell growth was independent of the ini­tial cell density of the culture. The generation time deter­mined in proliferating cultures during three days of expo­nential growth proved to be 17.9 ±0 .6 h. Addition of throm­bin (1 μg/ml culture medium) resulted in a reduction of the generation time to 16.1 ± 0 . 4 h.

Contact inhibition in confluent cultures

The contact inhibition of coronary endothelial cell growth already microscopically observed was further demonstrated by (methyl-3H)-thymidine incorporation studies. Applica­tion of autoradiographic techniques made it possible to de­termine labelling indices (percentage of labelled cells in a culture) which amounted to about 90% in growing, but only to about 5% in confluent cultures. Quantitative measure­ments of thymidine incorporation into highly polymerized DNA (TCA-insoluble) confirmed and extended the histo-chemical findings. Rates of DNA synthesis declined drasti­cally with the progression of confluency; thus minimal values were obtained in the stationary phase ( G 0 phase of growth, Fig. 5). As is evident from Figure 6, DNA content in the cul­

tures did not change any more when confluency was reached. However, total protein in the cultures still in­creased, most likely due to the synthesis of insoluble protein fractions, which formed a kind of layer underneath the con­fluent cells.

Ultrastructural appearance of cultured coronary endothelial cells

Freshly isolated cells formed numerous pseudopodia (Fig. 7a). The cytoplasm contained a great number of rather long and unbranched mitochondria, as well as vacuoles, granules and a great number of plasmalemmal vesicles. The shape of the nucleus appeared to be highly irregular with deep inden­tations. Weibel-Palade bodies were found only occasionally. Sometimes typical junctions could be detected which are known to be characteristic features of endothelial cells.

In contrast to dissociated and suspended cells, endothelial cells after one week in culture revealed a regular plasmalem­mal surface and an ovoid nucleus very much the same as in coronary endothelial cells in situ. Their cytoplasmic architec­ture (Fig. 7b) directly comparable to cells in situ (Fig. 2b) and to that of freshly isolated cells as shown above (Fig. 7a) remained rather unaltered during cultivation. In addition, amorphous basal lamina-like material could occasionally be detected underneath the cells of postconfluent cultures.

Scanning electron microscopic pictures of cultured endo­thelial cells revealed certain morphological changes during their attachment and proliferation. Immediately after seed­ing, the cells were characterized by a spherical shape (Fig. 8a), whereas after an initial phase of attachment between 2 and 10 min (Fig. 8b) "jelly-fish like" forms developed (Fig. 8c and d). Flattening of the cells after 10 to 20 h was accom­panied by the processes of DNA replication and cell division (Fig. 8e). In this phase all cells were characterized by numer­ous regularly formed granula accumulating especially in the perinuclear region. After about 48 h an unfenestrated and continuous endothelial monolayer had been formed in which some partial overlap of the individual cell boundaries be­came apparent (Fig. 8f).

Similar morphological features of proliferating endothelial cells not dissociated from the vascular bed could be demon­strated in dissected pieces of coronaries which were main­tained in organ culture. As is shown in Figure 9, the prolifer­ating and amoeboidically migrating endothelial cells in the immediate vicinity of the cutting line revealed similar mor­phological features as described above for isolated and cul­tured cells (Fig. 8).

Enzyme activities

In a first attempt to biochemically characterize coronary en­dothelial cells specific and total activities of 5'-nucleotidase and of nucleoside Phosphorylase were determined. These en­zymes were previously shown by histochemical techniques to be marker enzymes of the coronary endothelium in guinea pig hearts [46, 47]. As is evident from the data in Table I, both, the specific and total activities of the two enzymes proved to be rather high and almost identical in freshly iso­lated and cultured endothelial cells. In contrast, the corre­sponding activities of the same enzymes in ventricular myo­cardium were found to be very low.

292

Discussion

Isolation of pure endothelial cells from the coronary vessels is rendered difficult because of contaminations of the cell harvest with other cell types, such as pericardial and endo­cardial cells, pericytes, fibroblasts, and smooth muscle cells. Since all these cells are characterized by a high growth po­tential, long term tissue cultivation of coronary endothelial cells requires the complete elimination of all contaminants.

Several attempts were made in the past to isolate various types of cells from ventricular myocardium including coron­ary endothelial cells [33, 57, 27, 17, 28, 29]; however, the cell preparations obtained proved not to be homogeneous. Even a specifically developed disruption technique for myocardial tissue recently reported by Simionescu and Simionescu [51] which leaves most of the endothelial cells intact resulted in an endothelial cell harvest of only 85% purity.

The new methodological approach described in this paper is based on two subsequent steps: 1) a rather selective detach­ment of the endothelial cells from the intact vessel system in isolated guinea pig hearts by application of a standardized enzyme perfusion technique; 2) exclusion of the contaminat­ing cells (<2%) and of some tissue debris by Percoll density gradient centrifugation [41], a procedure, which proved opti­mal because of the strikingly low specific density of coronary endothelial cells (1.03 g/cm 3).

The isolated cells were characterized by various means in­cluding studies on their morphology, viability and specific enzymatic equipment. The morphological features of freshly isolated and cultured coronary endothelial cells revealed by phase microscopy as well as by scanning and transmission electron microscopy, resembled those observed in guinea pig hearts in situ. Moreover, they were very similar to those of endothelial cells of various other origins. The extremely rare occurrence of Weibel-Palade bodies in freshly isolated and cultured cells is of special interest. While these cytoplasmic constituents have been reported to be specific markers of the endothelium from a variety of blood vessels [56], they could not be detected in the endocardium of the frog [53], in bo­vine coronary endothelium [16] or in partially purified endo­thelial cells from rabbit hearts [51].

Due to the gentle isolation procedure applied the viability of the endothelial cells in culture proved to be satisfactory. Since replication and propagation of the endothelial cells were independent of the initial cell density in the cultures (similarly as reported for endothelial cells from bovine aorta [5]), it was possible to determine the generation time in ex­ponentially growing cultures, when contact inhibition of cell growth was negligible (G, phase of cell cycle). The low value of only 18 h for the endothelial cell cycle in vitro is in good agreement with findings concerning rapid repair mecha­nisms in various vessels in vivo after artificial lesion of the endothelium [48, 41]. In this connection it is of interest that thrombin in rather small concentrations was shown in our experiments to reduce the generation time from about 18 to about 16 h. Thus thrombin must be considered not only a mitogenic stimulator for human endothelial cells from the umbilical vein [58], but also for coronary endothelial cells. It appears likely that this function of thrombin is of general im­portance for a sufficiently quick repair of endothelial le­sions.

Postconfluent cultures of coronary endothelial cells were characterized by uniform monolayering of contact inhibited cells. This architecture of coronary endothelial cell cultures, which was also described for endothelial cell cultures from various other blood vessels [24, 55, 4, 15], appears to be di­rectly comparable with the arrangement of these cells in situ. All other cell types of the vessel wall hitherto successfully

Fig. 7. Transmission electron micrographs of freshly isolated and cultured coronary endothelial cells sectioned en face. — a. Isolated coronary endothelium. The two cells shown are joined by a typical junction (J). Microvil l i are present on the luminal and abluminal front. Most plasmalemmal vesicles are located within the cells. Pro­file of the nucleus (N) is irregular and deeply indented. — 9000 χ .— b. Coronary endothelial cells grown in tissue culture on a dextran bead (Cytodex, Pharmacia). The cytoplasmic architecture compares well with that of freshly isolated cells (a) or with that of coronary en­dothelial cells in situ (Fig. 2b). — Ε Endothelial cell.— C Dextran bead. — 3400 χ .

293

Fig. 8. Six scanning electron microscopic views o f coronary endo­thelial cells during attachment to their substratum. — a. 2 dissociated cells, immediately after seeding. —12000 χ . — b. A partially at­tached cell, 5 min after seeding.— 10000 χ . — c, d. "Jelly fish" like morphology of cells 30 and 120 min after seeding. — 8000 χ . — e. Dividing nuclei about 19 h after seeding, surrounded by an abundant number of granules. — 2900 χ . — f. Flattened cell after completion of the monolayer 48 h after seeding. Cell boundaries partly overlap those of the adjacent cells. —1800 χ .

grown in tissue culture (smooth muscle cells [45], fibroblasts [11]) have been shown to form multilayers of individual cells not subjected to contact inhibition.

Only a few biochemical marker substances of vascular en­dothelium have been identified as yet. These include clotting factor VIII antigen and blood group antigens which exist on the plasmalemmal surface of the human umbilical vein en­dothelium [24], as well as some ectoenzymes [50, 34]. Promis­ing candidates for a first biochemical characterisation of cul­tured endothelial cells from the coronaries seem to be 5'-nu­cleotidase and nucleoside Phosphorylase, since both these enzymes were shown by electron histochemical techniques to be prominent markers of the capillary endothelial cells of guinea pig hearts [46, 47]. The rather high specific and total activities of both enzymes in cultured coronary endothelial cells compared with the relatively low activities of these en­zymes in ventricular homogenates are in good agreement with the histochemical findings. Because the enzyme activi­ties were shown to be almost identical both in freshly iso­lated and in cultured coronary endothelial cells, it appears

reasonable to conclude that enzyme perfusion of isolated hearts for endothelial cell dissociation and the conditions of cell culture did not result in prolonged metabolic alterations of the cells.

Based on the assumption that total endothelial tissue con­tributes about 2% of the wet weight of the myocardium [51], about 20% of the total 5'-nucleotidase activity and almost 98% of the total nucleoside Phosphorylase activity in heart tissue can be considered to originate from the coronary endo­thelium. The remarkably high enzyme activities deserve par­ticular attention with respect to nucleotide metabolism in these cells. According to preliminary results [36-38] adenine nucleotide content of coronary endothelial cells proved to be extraordinarily high (about 15 μ π ι ο ^ ^ cells (wet weight)). In addition, these cells were shown to contain considerable amounts of adenosine. This vasoactive nucleoside known to originate from the breakdown of adenine nucleotides is as­sumed to be involved in the metabolic regulation of coronary blood flow [14, 48]. At present it is not yet clear whether the

Fig. 9. Scanning, electron micrograph of proliferating endothelial cells of a dissected coronary artery after 4 days in organ culture. — a. Overview showing in the left part the vascular surface, in the right part media and adventitia of the crossectioned vessel. — 1600 χ . —b . The marked area in a (5fold enlarged) reveals proliferating endothe­lial cells in the process of coating the crossectioned media of the ves­sel. The typical morphological features of these cells at different stages of the cell cycle closely resemble those observed on cultured coronary endothelial cells (Fig. 8). — 8000 χ .

294

peculiar distribution pattern of adenine nucleotides as well as of adenosine in coronary endothelial cells is of functional importance, for instance with respect to the interaction of en­dothelium with platelets as recently suggested [40], or regard­ing the metabolic regulation of coronary flow. Future studies devoted to a further elucidation of these possible interrela­tionships, however, will be greatly facilitated by using rou­tinely the techniques and procedures described in this pa­per.

Acknowledgements. One of the authors (J. S.) is indebted to the Fritz Thyssen Stiftung (1979/2/25) for generous financial support. The skilful technical assistance of Miss M . Arnhofer and of Mr. H. Sima are gratefully acknowledged. We are indebted to Prof. Dr. R. Wetz­stein for permission to use the scanning electron microscope of the Dept. of Anatomy, University of Munich. The assistance of Miss I . Schinko, Mrs. U . Hildebrandt, Mrs. A. Nekic and Mr. Chr. Grosse during the preparation of the specimens for scanning electron micro­scopy is greatly appreciated.

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